The present invention relates to certain thiophene derivatives and their use in the practice of medicine as allosteric modulators of adenosine receptors.
Adenosine (Ado) is an autocoid (or local hormone) that modulates numerous functions in the cardiovascular and other organ systems. The actions of Ado are mediated by at least four subtypes of cell surface receptors called A1, A2a, A2b, and A3. Because the ubiquity of adenosine receptors (AdoRs) throughout the body of a human, their indiscriminate activation may cause undesirable side effects. Therefore, new drug design approaches to achieve receptor and organ selectivity are needed.
The overall function of Ado appears to be the regulation of the balance between oxygen (or energy) supply and consumption (or work). Ado increases oxygen supply by causing vasodilation and decreases oxygen consumption or work by inhibiting cellular functions, e.g., slowing of heart rate. Consistent with this protective function, A1AdoR agonists, Ado uptake blockers and Ado deaminase inhibitors have been shown to reduce cellular damage and dysfunction during hypoxia and ischemia. This protective role of Ado and A1AdoR agonists has been shown in heart, brain, liver, and intestines. This and other potentially beneficial actions of Ado have led to increased interest in the development of Ado-related drugs targeted to ameliorate conditions such as myocardial ischemia and stroke.
However, the widespread expression of Ado receptors and the lack of sufficiently selective adenosine agonists have been a major impediment to the successful development of direct-acting AdoR agonists to exploit the cytoprotective properties of Ado. Therefore, other pharmacological approaches such as allosteric modulators of Ado may prove to be a valuable alternative to direct-acting Ado agonists and nucleoside uptake blockers. Such agents should selectively modulate the response to Ado in only those organs or localized areas of a given organ in which production of Ado is increased. Thus, allosteric modulators of Ado function should provide a more selective therapeutic effect than direct-acting AdoR agonists. Their action will be limited to times and locations at which significant release of Ado occurs so that systemic side effects would largely be avoided.
Allosteric modulation of the actions of Ado on the A1AdoR by several 2-amino-3-benzoylthiophenes on cultured cells, cardiac and brain preparations have been reported. The specificity of these compounds for A1AdoRs have also been demonstrated.
A number of compounds known to modulate the action of neurotransmitters, hormones and peptides bind at sites distinct from, but functionally linked to, the primary recognition site of the respective receptors. This form of interaction between two different ligands at the same receptor protein, which may result in modulation in the form of enhancement or inhibition of each others binding and function, is referred to as allosterism. Positive (enhancement) or negative (inhibition) allosterism is an important mechanism of action of drugs. For example, allosteric interactions between the GABA receptor and benzodiazepines, to atrial natriuretic factor (ANF) receptor and amiloride, the dextromethorphan binding site and ropizine, and the muscarinic receptor and gallamine have been described.
The compounds of the present invention have been found to be potent, yet selective allosteric modulators of AdoR agonists, and in some cases, AdoR antagonists.
Compounds useful as potent, yet selective allosteric modulators of adenosine receptors, with activity as AdoR agonists, and, in come cases AdoR antagonists, and methods of preparation and use thereof, are disclosed.
The compounds have the following general formulas IA and IB: 
wherein R2 is H, C(xe2x95x90O)R8;
R8 is H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl;
R3 and R4 are independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, hydroxy, alkylsulfonyloxy, arylsulfonyloxy, substituted arylsulfonyloxy, alkoxy, alkylthio, or arylthio;
or if R3 and R4 are both alkoxy or alkylthio, may form a 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, or 1,3-dithian-2-yl group;
or together R3 and R4 may form a carbonyl oxygen;
R5, R6, and R7 are independently H, alkyl, substituted alkyl, aryl, substituted aryl, halogen, hydroxy, nitro, amino, substituted amino, disubstituted amino, alkoxy, aryloxy, alkylthio, arylthio, sulfonamido, or substituted sulfonamido;
or together R5 and R6 or R6 and R7 may be CHxe2x95x90CHxe2x80x94CHxe2x95x90CH, such that they form a fused aromatic ring;
A and B are independently O, S, or Nxe2x80x94R8;
or A and B may independently represent a carbonxe2x80x94carbon single bond;
m and n are independently 0, 1, 2, or 3;
except that A and B cannot both represent a carbonxe2x80x94carbon single bond when m and n are both 0; and
X is CHxe2x95x90CH, CHxe2x95x90N, Nxe2x95x90CH, O, S, or Nxe2x80x94R8.
The compounds can be used in a method for cardioprotection, neuroprotection, pain management, reduction of free fatty acids, triglycerides, or glucose levels, adjunct therapy in diabetes, treatment of GI disorders, treatment of glaucoma; treatment of sleep disorders; treatment of cardiac disarrythmias (peroxysmal supraventricular tachycardia, treatment of congestive heart failure or treatment of inflammation.
The compounds can be used in a pharmaceutical formulation that includes a compounds of formulas IA or IB and one or more excipients. Various chemical intermediates can be used to prepare the compounds of formula IA or IB:
Not applicable.
The present application discloses compounds useful as potent, yet selective allosteric modulators of adenosine receptors, with activity as AdoR agonists, and in some cases, AdoR antagonists, and methods of preparation and use thereof.
The compounds can be used in a method for allosterically modulating adenosine receptors in a mammal, including a human. The methods involve administering an effective amount of a compound of formula IA or IB sufficient to moderate adenosine receptors to the mammal.
The compounds can be used in a pharmaceutical formulation that includes a compound of formula IA or IB and one or more excipients. Various chemical intermediates can be used to prepare the compounds of formula IA or IB.
As used herein the term xe2x80x9clower alkylxe2x80x9d means a monovalent radical, straight or branched chain, derived from the corresponding alkane having one to ten carbon atoms, i.e., methyl, ethyl, propyl, Isopropyl, n-butyl, sec-butyl, t-butyl, pentyl (all isomers), etc. Likewise, xe2x80x9clower alkylenexe2x80x9d means a divalent radical of the corresponding alkane. Further, as used herein, other moieties having names derived from alkanes, such as alkoxyl, alkanoyl, alkenyl, cycloalkenyl, etc when modified by xe2x80x9clower,xe2x80x9d have carbon chains of ten or less carbon atoms. In those cases where the minimum number of carbons are greater than one, e.g., alkenyl (minimum of two carbons) and cycloalkyl, (minimum of three carbons), it is to be understood that xe2x80x9clowerxe2x80x9d means at least the minimum number of carbons.
As used herein the term xe2x80x9camino acidxe2x80x9d means an alpha amino acid selected from those amino acids which naturally occur in proteins but without regard for specific stereochemical properties. The term xe2x80x9cprotected amino acidxe2x80x9d means an amino acid of which the alpha amino group has been converted to a less reactive moiety, but a moiety which can be converted back to the amino group with relative ease. The terms xe2x80x9camino acid residuexe2x80x9d and xe2x80x9camino acid moietyxe2x80x9d are use synonymously herein.
As used herein, the term xe2x80x9csubstituted alkylxe2x80x9d refers to an alkyl group, preferably of from 1 to 10 carbon atoms (xe2x80x9csubstituted lower alkylxe2x80x9d), having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, hydroxyamino, alkoxyamino, nitro, xe2x80x94SO-alkyl, xe2x80x94SO-substituted alkyl, xe2x80x94SO-aryl, xe2x80x94SO, heteroaryl, xe2x80x94SO2-alkyl, xe2x80x94SO?-substituted alkyl, xe2x80x94SO2-aryl, xe2x80x94SO2-heteroaryl, and mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclic amino, and unsymmetric di-substituted amines having different substituents selected from the group consisting of alkyl, aryl, heteroaryl and heterocyclic. As used herein, other moieties having, the prefix xe2x80x9csubstitutedxe2x80x9d are intended to include one or more of the substituents listed above.
As used herein, the term xe2x80x9calkoxyxe2x80x9d refers to the group xe2x80x9calkyl-Oxe2x80x94xe2x80x9d, where alkyl is as defined above. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
As used herein, the term xe2x80x9calkenylxe2x80x9d refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation. Preferred alkenyl groups include ethenyl (xe2x80x94CHxe2x95x90CH2), n-propenyl (xe2x80x94CH2CHxe2x95x90CH2), iso-propenyl (xe2x80x94C(CH3)xe2x95x90CH2), and the like.
As used herein, the term xe2x80x9calkynylxe2x80x9d refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.
As used herein, the term xe2x80x9cacylxe2x80x9d refers to the groups alkyl-C(O)xe2x80x94, substituted alkyl C(O)xe2x80x94, cycloalkyl-C(O)xe2x80x94, substituted cycloalkyl-C(O)xe2x80x94, aryl-C(O)xe2x80x94, substituted aryl-C(O)xe2x80x94, heteroaryl-C(O)xe2x80x94 and heterocyclic-C(O)xe2x80x94 where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term xe2x80x9cacylaminoxe2x80x9d refers to the group xe2x80x94C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or heterocyclic, wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term xe2x80x9carylxe2x80x9d refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring, (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, xe2x80x94SO-alkyl, xe2x80x94SO-substituted alkyl, xe2x80x94SO-aryl, xe2x80x94SO-heteroaryl, xe2x80x94SO2-alkyl, SO2-substituted alkyl, xe2x80x94SO2-aryl, xe2x80x94SO2-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
As used herein, the term xe2x80x9ccycloalkylxe2x80x9d refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
As used herein, the terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refer to fluoro, chloro, bromo and iodo and preferably is either fluoro or chloro.
As used herein, the term xe2x80x9cheteroarylxe2x80x9d refers to an aromatic carbocyclic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).
Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, xe2x80x94SO-alkyl, xe2x80x94SO-substituted alkyl, xe2x80x94SO-aryl, xe2x80x94SO-heteroaryl, xe2x80x94SO2-alkyl, _SO2-substituted alkyl, xe2x80x94SO2-aryl, xe2x80x94SO2-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thloalkoxy. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).
xe2x80x9cHeterocyclexe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d refers to a monovalent saturated or unsaturated carbocyclic group having a single ring or multiple condensed rings, from 1 to 15 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups are optionally substituted with 1 to 5 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, trihalomethyl, and the like. Such heterocyclic groups can have a single ring or multiple condensed rings.
As to any of the above groups that contain 1 or more substituents, it is understood, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d refers to pharmaceutically acceptable salts of a compound of Formulas IA or IB, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can be used as the pharmaceutically acceptable salt.
The term xe2x80x9cprotecting groupxe2x80x9d or xe2x80x9cblocking groupxe2x80x9d refers to any group which when bound to one or more hydroxyl, amino or carboxyl groups of the compounds (including intermediates thereof such as the aminolactams, aminolactones, etc.) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, amino or carboxyl group. Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), and the like which can be removed by conventional conditions compatible with the nature of the product.
The compounds of the present invention have the following general formulas IA and IB: 
wherein R2 is H, C(xe2x95x90O)R8;
R8 is H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl;
R3 and R4 are independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, hydroxy, alkylsulfonyloxy, arylsulfonyloxy, substituted arylsulfonyloxy, alkoxy, alkylthio, or arylthio;
or if R3 and R4 are both alkoxy or alkylthio, may form a 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, or 1,3-dithian-2-yl group; or together R3 and R4 may form a carbonyl oxygen;
R5, R6, and R7 are independently H, alkyl, substituted alkyl, aryl, substituted aryl, halogen, hydroxy, nitro, amino, substituted amino, disubstituted amino, alkoxy, aryloxy, alkylthio, arylthio, sulfonamido, or substituted sulfonamido;
or together R5 and R6 or R6 and R7 may be CHxe2x95x90CHxe2x80x94CHxe2x95x90CH, such that they form a fused aromatic ring;
A and B are independently O, S, or N-R8;
or A and B may independently represent a carbonxe2x80x94carbon single bond;
m and n are independently 0, 1, 2, or 3;
except that A and B cannot both represent a carbonxe2x80x94carbon single bond when m and n are both 0; and
X is CHxe2x95x90CH, CHxe2x95x90N, Nxe2x95x90CH, O, S, or Nxe2x80x94R8.
Those skilled in the art of organic chemistry will appreciate that reactive and fragile functional groups often must be protected prior to a particular reaction, or sequence of reactions, and then restored to their original forms after the last reaction is completed. Usually groups are protected by converting them to a relatively stable derivative. For example, a hydroxyl group may be converted to an ether group and an amino converted to an amide or carbamate. Methods of protecting and de-protect, also know as xe2x80x9cblockingxe2x80x9d and xe2x80x9cde-blocking,xe2x80x9d are well know and widely practiced in the art, e.g., see T. Green, Protective Groups in Organic Synthesis, John Wiley, New York (1981) or Protective Groups in Organic Chemistry, Ed. J. F. W. McOmie, Plenum Press, London (1973).
Representative example compounds of the present invention include:
Synthesis of Compounds
Compounds of formula IA may be conveniently prepared according to Scheme 1. A compound of formula (II) or (III) is reacted with a compound of formula (IV) in the presence of elemental sulfur and morpholine at approximately 70xc2x0 C., for about one hour, then at approximately 20 to 25xc2x0 C. for a period of about 20 hours to yield a compound of formula (IA). 
Compounds of formula (IB) may be conveniently prepared according to Scheme 2. In a manner analogous to that used for preparing compounds of formula (IA), 2-butanone is reacted with a compound of formula (II) or (III) in the presence of elemental sulfur and morpholine at approximately 70xc2x0 C., for about one hour, then at approximately 20 to 25xc2x0 C., for a period of about 20 hours, to afford a compound of formula (IB). 
Compounds of formula (II) or (III) may be conveniently prepared according to Scheme 3 by treating compounds of formula (V) or (VI) with bromine in glacial acetic acid at approximately 20 to 25xc2x0 C. for approximately two hours to provide intermediates of formula (VII) or (VIII). These intermediates in a protic solvent, such as ethanol, may then be treated with an aqueous solution of potassium cyanide at approximately 20 to 25xc2x0 C. for about 20 to 24 hours to afford compounds of formula (II) or (III). 
Compounds of formula (IV), (V), or (VI) are commercially available or may be prepared by methods known in the literature.
Utility
The compounds can be used for:
Protection against hypoxia and/or ischemia induced injuries (e.g., stroke, infarction);
Treatment of adenosine-sensitive cardiac arrhythmias;
antinociception (i.e., analgesics);
anticonvulsants;
cardioprotection, short term (e.g., prior to percutaneous angioplasty (PTDA), angioplasty, and cardiac surgeries) and long term (prevention of myocardial infarction, especially in high risk patients, reduction of infarct damage, especially in high risk patients);
treatment of congestive heart failure;
neuroprotection: stroke prevention, stroke treatment, treatment of Alzheimer""s disease and treatment of epilepsy;
pain management generally, including different forms of neuropathic pain, e.g., diabetic neuropathy, post herpetic neuralgia;
antilipid uses: reduction of free fatty acids, triglycerides, glucose;
adjunct therapy in diabetes, including, insulin dependent and non-insulin dependent diabetes mellitus: stimulation of insulin secretion from the pancreas, increase in tissue sensitivity to insulin;
treatment of GI disorders such as diarrhea, irritable bowel disease, irritable bowel syndrome, irritable bladder, incontinence such as urge incontinence;
treatment of glaucoma;
treatment of sleep diorders, such as sleep apnea;
treatment of cardiac disarrythmias (peroxysmal supraventricular tachycardia;
use in combination with anesthesia for post surgical pain; treatment of inflammation;
diagnostic uses, for example, to determine the presence of one or more of the above described medical conditions, or in a screening assay to determine the effectiveness of other compounds for bindinor to the Al Ado receptor (i.e., through competitive inhibition as determined by various
binding assays); and
Other indications for which A1AdoR agonists are used.
The amount of compound of the present invention required to be effective as an allosteric modulator of an adenosine receptor will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, the nature of the formulation, the mammal""s body weight, surface area, age and general condition, and the particular compound to be administered. However, a suitable effective dose is in the range of about 0.1 xcexcg/kg to about 10 mg/kg body weight per day, preferably in the range of about 1 mg/kg to about 3 mg/kg per day.
The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary. For example, for a 75 kg mammal, a dose range would be about 75 mg to about 220 mg per day, and a typical dose would be about 150 mg per day. If discrete multiple doses are indicated, treatment might typically be 50 mg of a compound of the present invention given 3 times per day.
Biological Assays
Chinese hamster ovary cells expressing human recombinant A1 adenosine receptors (CHO:huA1 cells) at a density of approximately 8000 fmol/mg protein were prepared as previously described (Kollias-Baker et al., (1997), J. Pharmacol. Exp. Ther. 281: 761-768) and aliquots of the cells at low passage numbers were frozen and stored in liquid nitrogen. When compounds were tested, an aliquot of CHO:huA1 cells were rapidly thawed after removal from liquid nitrogen, then grown in Ham""s F12 culture medium with 10% fetal bovine serum and 0.5 mg/mL of antibiotic G-418 (Shryock, Ozeck, and Belardinelli (1998), Mol. Pharmacol 53: 886-893). Cells were passaged thrice weekly. Aliquots of cells were placed into 12-well plates with culture medium, serum, and antibiotic for 48 hours, by which time the cells had grown to a confluent monolayer.
Allosteric enhancement was measured as the action of a test compound at different concentrations (0.01, 0.1, 1 and 10 xcexcM) to reduce the cAMP content of CHO:huA1 cells. To initiate an experiment, growth medium was removed from the 12-well plates and cells were washed once with warm Hanks"" buffered saline. The wash solution was then removed and replaced with fresh Hanks"" solution containing forskolin (1 xcexcM), rolipram (20 xcexcM), N6-cyclopentyladenosine (CPA, 0.01 nM), adenosine deaminase (2 U/mL), and the test compound. Forskolin was used to stimulate the activity of adenylyl cyclase, rolipram to inhibit cAMP phosphodiesterase, adenosine deaminase to degrade endogenous adenosine, and CPA to cause a small increase of the number of activated adenosine receptors. After 6 min of incubation at 36xc2x0 C. in the presence of test compound, the incubation solution was removed and hydrochloric acid (final concentration 50 mM) was added to terminate drug action.
The content of cAMP in acidified extracts of cells was determined by radioimmunoassay as previously described (Kollias-Baker et al., (1997), J. Pharmacol. Exp. Ther. 281: 761-768). Because the magnitude of the effects of allosteric enhancers on CHO:huA1 cells changed subtly with passage number and differed slightly among different aliquots of cells, the actions of the test compounds and the action of a reference compound (PD 81,723) were assessed in each experiment. The effect of each test compound on cAMP content is presented in Table 1 as a percentage of the value of cAMP content in the absence of drug (control, 100%). Each value is a mean+standard error of 6 determinations in each of the number of experiments indicated in the xe2x80x9cnxe2x80x9d column of the table.
Radioligand Binding Assays Results
Methods for detecting specific macromolecular species, such as proteins, drugs, and polynucleotides, have proven to be very valuable analytical techniques in biology and medicine, particularly for characterizing the molecular composition of normal and abnormal tissue samples and genetic material. Many different types of such detection methods are widely used in biomedical research and clinical laboratory medicine. Examples of such detection methods include: immunoassays, immunochemical staining for microscopy, fluorescence-activated cell sorting (FACS), nucleic acid hybridization, water sampling, air sampling, and others. Typically, a detection method employs at least one analytical reagent that binds to a specific target macromolecular species and produces a detectable signal. These analytical reagents typically have two components: (1) a probe macromolecule, for example, an antibody or oligonucleotide, that can bind a target macromolecule with a high degree of specificity and affinity, and (2) a detectable label, such as a radioisotope or covalently-linked fluorescent dye molecule. In general, the binding properties of the probe macromolecule define the specificity of the detection method, and the detectability of the associated label determines the sensitivity of the detection method. The sensitivity of detection is in turn related to both the type of label employed and the quality and type of equipment available to detect it.
For example, radioimmunoassays (RIA) have been among the most sensitive and specific analytical methods used for detecting and quantitating biological macromolecules. Radioimmunoassay techniques have been used to detect and measure minute quantities of specific analytes, such as polypeptides, drugs, steroid hormones, polynucleotides, metabolites, and tumor markers, in biological samples. Radioimmunoassay methods employ immunoglobulins labeled with one or more radioisotopes as the analytical reagent. Radiation (alpha, beta, or gamma) produced by decay of the attached radioisotope label serves as the signal which can be detected and quantitated by various radiometric methods.
The term xe2x80x9clabeled analogxe2x80x9d is further understood to encompass compounds which are labeled with radioactive isotopes, such as but not limited to tritium (3 H), carbon (14C), iodine (1251), phosphorus (31P, 32P, 33P), sulfur (35S) or otherwise labeled (e.g. fluorescently). Less common, but also known in the art, is the use of radio labels of the positron emitting radionuclides 11C, 13N, 15O, 18F or 76Br. Also known in the art, but less common, is the use of stable isotopes, such as deuterium (2H) and 13C that are detected by magnetic resonance imaging or mass spectrometry. The compounds of this invention may be labeled or derivatized, for example, for kinetic binding experiments, for further elucidating metabolic pathways and enzymatic mechanisms, or for characterization by methods known in the art of analytical chemistry.
Formulations
Formulations of the present invention for medical use comprise an active compound, i.e., a compound of formula (IA), (IB) or (IC), together with an acceptable carrier thereof and optionally other therapeutically active ingredients. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of formula (IA), (IB) or (IC) together with a pharmaceutically acceptable carrier thereof.
The formulations include, but are not limited to, those suitable for oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred are those suitable for oral or parenteral administration.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound;
as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients. Such accessory ingredients) may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.
Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, 5% dextrose in distilled water or saline. Useful formulations also comprise concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration above.
Topical formulations include ointments, creams, gels and lotions which may be prepared by conventional methods known in the art of pharmacy. In addition to the ointment, cream gel, or lotion base and the active ingredient, such topical formulation may also contain preservatives, perfumes, and additional active pharmaceutical agents.
In addition to the aforementioned ingredients, the formulations of this invention may further include one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.